title: Combination of Epstein-Barr virus nuclear antigen 1, 3 and lytic antigen BZLF1 peptide pools allows fast and efficient stimulation of Epstein-Barr virus-specific T cells for adoptive immunotherapy.
abstract: BACKGROUND
  Epstein-Barr virus (EBV) infection is a major cause of morbidity following hematopoietic stem cell transplantation. EBV-infected B cells may not respond to rituximab treatment and may lead to a life-threatening post-transplantation lymphoproliferative disorder. Adoptive cellular immunotherapy using EBV-lymphoblastoid cell lines (LCL) as stimulating antigen has proved effective in restoring specific immunity. However, EBV presents several immunodominant antigens, and developing a swift and effective clinical-grade immunotherapy relies on the definition of a Good Manufacturing Practices (GMP) universal stimulating antigen.
  
  
  METHODS
  Peripheral blood mononuclear cells (PBMCs) from six donors with a cellular immune response against EBV were immunoselected after stimulation with a new EBV antigen associated with an EBNA3 peptide pool.
  
  
  RESULTS
  After immunoselection, a mean of 0.53 ± 0.25 × 10⁶ cells was recovered consisting of a mean of 24.77 ± 18.01% CD4⁺-secreting interferon (IFN)-γ and 51.42 ± 26.92% CD8⁺-secreting IFN-γ. The T memory stem cell sub-population was identified. EBV-specific T cells were expanded in vitro, and their ability to secrete IFN-γ and to proliferate after re-stimulation with EBV antigen was confirmed. A specific lysis was observed against autologous target cells pulsed with EBV peptide pools (57.6 ± 11.5%) and against autologous EBV-LCL (18.3 ± 7.3%). A mean decrease of 94.7 ± 3.3% in alloreactivity against third-party donor mononuclear cells with EBV-specific T cells was observed compared with PBMCs before selection.
  
  
  CONCLUSIONS
  Our results show that a combination of peptide pools including EBNA3 is needed to generate EBV-specific T cells with good specific cytotoxicity and devoid of alloreactivity, but as yet GMP grade is not fully achieved.
authors.affiliation: CHU de Nancy, Unité de Thérapie Cellulaire et Tissus, Vandoeuvre-lès-Nancy, France; CNRS, UMR 7365 et FR 3209, Faculté de Médecine, Université de Lorraine, Vandoeuvre-lès-Nancy, France.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: CHU de Nancy, Unité de Thérapie Cellulaire et Tissus, Vandoeuvre-lès-Nancy, France; CNRS, UMR 7365 et FR 3209, Faculté de Médecine, Université de Lorraine, Vandoeuvre-lès-Nancy, France.
authors.forename: Lamia
authors.initials: L
authors.lastname: Aïssi-Rothe
authors.affiliation: CHU de Nancy, Epidémiologie et Evaluation Cliniques, Vandoeuvre-lès-Nancy, France.
authors.forename: Jean Marc
authors.initials: JM
authors.lastname: Virion
authors.affiliation: CHU de Nancy, Laboratoire d'Immunologie et Plateforme Nancytomique, Vandoeuvre-lès-Nancy, France.
authors.forename: Marcelo
authors.initials: M
authors.lastname: De Carvalho Bittencourt
authors.affiliation: CHU de Nancy, Unité de Thérapie Cellulaire et Tissus, Vandoeuvre-lès-Nancy, France.
authors.forename: Neslihan
authors.initials: N
authors.lastname: Ulas
authors.affiliation: CHU de Nancy, Laboratoire d'Immunologie et Plateforme Nancytomique, Vandoeuvre-lès-Nancy, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Audonnet
authors.affiliation: CHU de Nancy, UTMA, Vandoeuvre-lès-Nancy, France.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Salmon
authors.affiliation: CHU de Nancy, UTMA, Vandoeuvre-lès-Nancy, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Clement
authors.affiliation: CHU de Nancy, Laboratoire de Virologie, Vandoeuvre-lès-Nancy, France.
authors.forename: Veronique
authors.initials: V
authors.lastname: Venard
authors.affiliation: CHU de Nancy, Laboratoire de Virologie, Vandoeuvre-lès-Nancy, France.
authors.forename: Helène
authors.initials: H
authors.lastname: Jeulin
authors.affiliation: CHU de Nancy, Unité de Thérapie Cellulaire et Tissus, Vandoeuvre-lès-Nancy, France; CNRS, UMR 7365 et FR 3209, Faculté de Médecine, Université de Lorraine, Vandoeuvre-lès-Nancy, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Stoltz
authors.affiliation: CHU de Nancy, Unité de Thérapie Cellulaire et Tissus, Vandoeuvre-lès-Nancy, France; CNRS, UMR 7365 et FR 3209, Faculté de Médecine, Université de Lorraine, Vandoeuvre-lès-Nancy, France.
authors.forename: Veronique
authors.initials: V
authors.lastname: Decot
authors.affiliation: CHU de Nancy, Unité de Thérapie Cellulaire et Tissus, Vandoeuvre-lès-Nancy, France; CNRS, UMR 7365 et FR 3209, Faculté de Médecine, Université de Lorraine, Vandoeuvre-lès-Nancy, France; Faculté de Pharmacie, Département de Microbiologie-Immunologie, Université de Lorraine, Nancy Cedex, France. Electronic address: d.bensoussan@chu-nancy.fr.
authors.forename: Danièle
authors.initials: D
authors.lastname: Bensoussan
mesh_terms: D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000821:virology; D020031:Epstein-Barr Virus Infections / Q000276:immunology / Q000628:therapy; D019309:Epstein-Barr Virus Nuclear Antigens / Q000276:immunology / Q000627:therapeutic use*; D018380:Hematopoietic Stem Cell Transplantation / Q000009:adverse effects; D004854:Herpesvirus 4, Human / Q000276:immunology; D006801:Humans; D007111:Immunity, Cellular / Q000276:immunology; D016219:Immunotherapy, Adoptive; D013601:T-Lymphocytes / Q000378:metabolism* / Q000821:virology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000821:virology; D015534:Trans-Activators / Q000276:immunology / Q000627:therapeutic use*
chemical_list: C063676:BZLF1 protein, Herpesvirus 4, Human; D019309:Epstein-Barr Virus Nuclear Antigens; D015534:Trans-Activators
keywords: EBV-specific T cells; Epstein-Barr virus infection; T memory stem cells (T(SCM)); adoptive immunotherapy; post-transplantation lymphoproliferative disorder (PTLD)